WO1992001383A1

WO1992001383A1 - Process for the production of compositions containing biosynthetic pesticidal products and use of the compositions obtained

Info

Publication number: WO1992001383A1
Application number: PCT/DK1991/000213
Authority: WO
Inventors: René Detroz; Michel Caprasse; Lucien Charmoille
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1990-07-23
Filing date: 1991-07-18
Publication date: 1992-02-06
Anticipated expiration: 1993-01-23
Also published as: AU8296491A; EP0499629A1; JPH05501723A; PL294568A1; HU9201365D0; CS85292A3; CA2066664A1; HUT65725A; BR9105844A; BE1004492A3

Abstract

The invention relates to a process for the production of compositions containing a biosynthetic pesticide product from microorganisms of the Bacillaceae family and at least tannic acid. The compositions obtained are effective against insects.

Description

Process for the production of compositions containing biosynthetic pesticidal products and use of the compositions obtained

The present invention relates to a process for the production of compositions containing biosynthetic pesticidal products obtained from microorganisms of the

Bacillaceae family and the use of the compositions obtained.

The process for obtaining biosynthetic pesticidal products from microorganisms generally comprises the following principal steps:

- fermentation of the microorganism to obtain a culture,^" - collection of this culture and

- formulation of the product in the form of a paste, powder, granulate or cream.

The collection of the culture is a delicate step in this process; in fact this step, which consists principally in separating the culture in the form of biomass and the culture liquid, is one of the causes of loss of product and therefore of the poor yield generally- obtained.

In addition, the active material of the pesticidal product is a sensitive compound by virtue of its biological structure which is easily denatured if certain conditions are not respected, such as, in particular, the temperature, the pH, the solvents and the additives. The poor yields observed during harvesting are due in particular to the poor separation of numerous constituents of the culture, which is viscous to a greater or lesser extent, to mechanical effects in the separation which cause the degradation of the active material of the pesticidal product, and therefore involve a fall in its biological activity, to the addition of adjuvants which affect the stability of the composition and/or its physical appearance, such as the fluidity of the paste, the separation of the paste or of the cream during storage, and the dispersion of the composition in water. In addition, an apparent fall in the biological activity is likewise observed, in particular when the composition is formulated in the form of paste. This formulation is advantageous in certain cases because it avoids the drying step which degrades the product to a greater or lesser extent and which is costly but is necessary for long-term preservation.

In fact, during the calculation of the extent of the biological activity, a deficit of 30 to 50% between the biological activity of the culture and that of the paste is observed, whereas a lower deficit between the biological activity of the powder obtained from drying this paste and that of the culture is observed. The biological activity of the paste is therefore apparently not completely available or accessible. The present invention has the aim of obtaining compositions which no longer have these disadvantages. In particular, it has the aim of obtaining compositions having a biological activity which is as high as possible and obtained in an economic fashion, that is to say avoiding the losses in yield at all levels, as well as from the point of view of quantity as of quality, and of biological activity, although being stable and having a satisfactory physical appearance. In addition, it is observed that the compositions according to the invention have a particularly high biological activity.

To this end, the invention relates to a process for the production of compositions containing biosynthetic pesticidal products obtained from micro¬ organisms of the Bacillaceae family and at least one tannin or a tannin derivative, this process comprising the following steps:

1) in a first step, the microorganism is cultured in a culture medium to obtain an abundant culture of this microorganism, 2) in a second step, a quantity of tannin or a tannin derivative is added to the culture obtained,

3) in a third step, the water is separated from the culture containing the tannin or the tannin derivative in order to obtain a paste, and

4) in a fourth step, the formulation of the paste thus obtained is carried out.

The preferred process according to the invention is that where tannic acid is added to the culture during the second step.

The biosynthetic pesticides obtained from the Bacillaceae family can be found in the concentrated compositions in very different forms. Thus, they can be present in the form of the organisms themselves at any stage of their development, including possible vegetative forms, as they are, in association with their culture medium, in a completely or partially lysed form, in a completely or partially sporulated form, in a form having partially or completely released the spores by various means such as bacterial autolysis, or in the form of products which are spontaneously excreted by the organisms, such as exotoxins, in the form of products which are extracted from these organisms, such as endotoxins, by any method known per se, involving or not involving lysis of the organism concerned, in the form of products possibly liberated by the organisms in the course of certain steps of their development (crystals or endotoxins associated or not associated with spores) or alternatively in several of these forms simultaneously. These different forms may possibly be associated with residues from the culture medium. Good results have been obtained with the mixture containing the spores, the crystals or the associated endotoxins and, possibly, exotoxins which are formed spontaneously during autolysis of the bacterium at the end of sporulation. Such a mixture may contain, besides the spores, the crystals associated or not associated with the spores and the possible exotoxins, cells or cell debris as well as residual solid products of the nutrient medium used during culture.

As microorganisms of the Bacillaceae family highly suitable in general as a source of biosynthetic pesticides, the microorganisms of the genus Bacillus such as Bacillus thuringiensis, Bacillus sphaericus, Bacillus popilliae, Bacillus cereus, Bacillus larvae, Bacillus lentimorbus, Bacillus fribourgensis and Bacillus penetrans (Pasteuria) are used.

Good results have been obtained with Bacillus sphaericus and Bacillus thuringiensis of all serotypes, these including bacteria resulting from genetic transformation, and more particularly Bacillus sphaericus of serotypes la, 2a2b, 2a2c, 5a5b, 6, 25, 26a26b, 26a26c and Bacillus thuringiensis of serotypes 1, 3a, 3a3b, 4, 6, 7, 8, 8a8b, 9 , 10 and 14. The best results have been obtained with Bacillus sphaericus of serotypes 5a5b and 25 and Bacillus thuringiensis of serotypes 1 (thuringiensis variety), 3a3b (kurstaki variety), 6 (subtoxicus and entomocidus varieties), 7 (aizawai variety), 8a8b (morrisoni variety) and 14 (israelensiε variety) . The various types of Bacillus thuringiensis and of Bacillus sphaericus can be used in the compositions according to the invention in the form of the microorganisms themselves, as such or lysed, of their spores, of endotoxins or of crystals associated or not associated with their spores or in several of these forms. These different forms may possibly be associated with residues of the culture medium. Good results have been obtained by using the mixture containing spores and the associated crystals which is formed spontaneously during the autolysis of the bacterium at the end of sporulation. Such a mixture may contain, besides the spores and the crystals associated or not associated with the spores, cells or cell debris as well as residual solid products of the nutrient medium used during culture.

The compositions obtained according to the invention can be present in the form of a suspension of solid particles of biosynthetic pesticidal products in a liquid or in the form of a solid such as a powder or a^' granulate.

The compositions obtained according to the invention may in addition contain various other additives of which the nature and the function depend on the use of these compositions and which are known by the person skilled in the art.

The compositions obtained according to the invention can likewise contain residues from the production of the biosynthetic pesticide.

When the compositions obtained according to the invention are present in the form of suspensions in a liquid, these suspensions in general contain from 10 to 45% by weight and most frequently from 15 to 40% by weight of biosynthetic pesticidal products. The liquid must be inert with respect to the biosynthetic pesticides and it must especially not deactivate them or provoke their coagulation or any other physical alteration. The liquid can be formed by a single solvent or by a mixture of solvents. In general, the solvent contains water.

Good results have been obtained when the solvent contains at least 80% and most frequently at least 95% by weight of water. In general, the liquid present in the composition according to the invention is formed essentially of water.

In general, during the second step, the necessary quantity of tannin or of tannin derivatives is between 0.05 and 4% by weight of the culture, usually between 0.1 and 3% and preferably between 0.25 and 2%. The third step, which consists in separating the water from the culture, is carried out by customary techniques for the separation of water such as centrifugation, ultrafiltration, precipitation by addition of a non-solvent such as acetone, filtration or flocculation so as to obtain a paste. Preferably, it is carried out by centrifugation and/or ultrafiltration.

The other possible additives are then added to this paste during formulation.

In the case of Bacillus thuringiensis and Bacillus sphaericus, the culture medium is taken, for- example, at the end of the sporulation phase.

Another process consists in separating fractions rich in endotoxins or in spores or in these two products simultaneously from the culture medium of the microorganisms generating biosynthetic pesticidal products at the end of the sporulation phase. These fractions are then treated according to the same techniques as those used for the treatment of the pastes. To obtain compositions in the form of dry powders, the paste thus obtained is dried by conventional techniques such as spray drying, freeze drying or drying in a drum so as to obtain solid particles in general containing less than 12% and preferably less than 7% of water.

Another technique consists in adding the other possible additives to the paste before drying it and in submitting the mixture to drying according to one or other of the techniques indicated above. To obtain the compositions according to the invention in the form of suspensions, the solid pesticide particles can be dispersed in a liquid phase which preferably contains water and which additionally contains the tannin or the tannin derivative as well as the other possible additives, to form a cream.

The present invention likewise relates to a process for the use of the compositions.

The compositions according to the invention can be used as pesticides and more particularly as agents to eliminate the insects at any stage of their development. To do this, they are sprayed onto the infested surfaces, plants, rivers, streams and any stretch of water according to all methods known per se such as manual spraying, mechanical spraying and more particularly aerial spraying. They can in particular be sprayed onto surfaces infested by insects and more particularly by Lepidopter, Dipter, Coleopter, Aphanipter, Orthopter, Isopter and Homopter.

The doses to be used depend on the biosynthetic - 1 -

pesticide used and of the target to be eliminated.

In the case of Bacillus thuringiensis and Bacillus sphaericus, from 50 to 5000 g of solid particles per hectare are in general sprayed. The compositions according to the invention can be sprayed as such when they are present in the form of suspensions. They can likewise be dispersed, diluted or emulsified in a diluent such as water or an organic diluent. In this case, they are diluted by 1 to 300 times, usually 2 to 120 times and preferably 4 to 60 times their volume of diluent.

The examples which follow serve to illustrate the invention. EXAMPLE 1

450 g of soya flour containing approximately 52% of proteins, 250 g of hydrolysed starch, 20 g of KH₂P0_<,, 20 g of ₂HP0₄, 3.2 g of MgS0.7H₂0, 0.12 g of MnS0^.H₂0, 0.14 g of ZnS0₄.7H₂0, 0.2 g of Fe₂(S0 ₃, 1.5 g of CaCl₂.H₂0 and 101 of water are introduced into a sterilisable 15 1 BIOLAFFITE fer enter equipped with a stirrer, a pH probe, an oxygen probe and an aeration system.

This culture medium is mixed until it is homogeneous; the pH of this medium is brought to 7.

This culture medium is sterilised at 120°C for 30 minutes. The culture medium is cooled to bring it to a temperature of 30°C and 1 1 of a culture of Bacillus thuringiensis serotype 14 (israelensis variety) in the growth phase is introduced into the fermenter in a sterile manner. The fermenter is stirred at 450 revolutions per minute and aerated at 0.4 wm (volume of air per volume of medium per minute) .

After 32 hours, the Bacillus thuringiensis is present in the form of a partially lysed sporulated bacterium. This culture contains 1.1 x 10⁹ spores per ml, its pH is 7.5 and its dry matter is 3 g per 1.

The insecticidal activity of this culture is measured with the aid of Aedes aegypti according to the biological titration method of H. de Barjac and - 8 -

I. Larget, WHO-VBC-79.744. It amounts to 300 U/ml +/- 20%.

2 parts per thousand of powdered citric acid are added to the product thus obtained. The pH of this product is adjusted to 4 by addition of phosphoric acid.

5 g of tannin, trademark TA IN PB5 from the company PHARMACHEMIC, containing 99% of tannic acid is introduced into 500 g of the product thus obtained. The insecticidal activity of the product obtained is equal, with the exception of errors of measurement, to that of the culture, and the acids and the tannin added are therefore not toxic to the target insects.

The product is then concentrated by centrifugation to give a paste containing approximately 12% of dry matter.

This paste has an insecticidal activity of 2800 U/mg +/- 20%.

This paste is fluid and stable in the course of time.

EXAMPLE 2 R (comparison)

A culture of Bacillus thuringiensis serotype 14 is produced as in Example 1. A paste identical to that of Example 1 is then prepared with the exception of the addition of tannin.

The paste obtained has an insecticidal activity of 1500 U/mg +/- 20%.

The results compared between Example 1 according to the invention and comparison Example 2R demonstrate that the incorporation of tannic acid into the culture before centrifugation allows harvesting to be facilitated, the centrifugation yield to be improved, and the active material of the product to be made available. Example 3R (comparison) A culture of Bacillus thuringiensis serotype 14 is produced as in Example 1. A paste identical to that of Example 1 is then prepared with the exception of the addition of tannin which is replaced by the addition of 0.1% of silica, trademark AEROSIL R 972 silica from the Company DEGUSSA .

The insecticidal activity of the paste obtained amounts to 1300 U/mg +/- 20%. The paste is compact and does not disperse in water.

Claims

C L A I M S

1. Process for the production of compositions containing biosynthetic pesticidal products obtained from microorganisms of the Bacillaceae family and at least one tannin or a tannin derivative, characterised in that it comprises the following steps:

1) in a first step, the microorganism is cultured in a culture medium to obtain an abundant culture of this microorganism, 2) in a second step, a quantity of tannin or a tannin derivative is added to the culture obtained, 3) in a third step, the water is separated from the culture containing the tannin or the tannin derivative in order to obtain a paste, and 4) in a fourth step, the formulation of the paste thus obtained is carried out.

2. Process according to Claim 1, characterised in that tannic acid is added in the second step.

3. Process according to any one of the preceding claims, characterised in that the microorganisms cultured are microorganisms of the Bacillus thuringiensis or Bacillus sphaericus types.

4. Process according to any one of the preceding claims, characterised in that in the second step a quantity of tannin or of tannin derivative of between 0.05 and 4% by weight of the culture is added to the culture.

5. Process to eliminate insects, characterised in that the compositions obtained according to any one of Claims 1 to 4 are sprayed onto the infested surfaces.